Blood coagulation factor VIII which is absent or defective in patients with hemophilia A, a severe congenital bleeding disorder, functions as a cofactor in the Xase complex involved in the anionic phospholipid surface-dependent conversion of Factor X to Factor Xa by Factor IXa (Non-Patent Document 1). Factor VIII is protected and stabilized by vWF which circulates as a complex with this cofactor (Non-Patent Document 2). The factor is synthesized as a single-chain multi-domain molecule (A1-A2-B-A3-C1-C2) consisting of 2,332 amino acid residues with a molecular weight up to 300 kDa (Non-Patent Documents 3 and 4), and processed into a series of metal ion-dependent heterodimers by cleavage at the B-A3 junction, generating a heavy chain consisting of the A1 and A2 domains, and heterogeneous fragments of partially proteolyzed B domains, and a light chain consisting of the A3, C1, and C2 domains that binds with the heavy chain (Non-Patent Documents 3 to 5).
Factor VIII is converted into activated blood coagulation factor VIII (also referred to as activated blood coagulation factor VIII or Factor VIIIa) by limited proteolysis with thrombin or Factor Xa (Non-Patent Document 6). Cleavage of the heavy chain at Arg372 and Arg740 generates 50-kDa A1 and 40-kDa A2 subunits. On the other hand, cleavage of the 80-kDa light chain at Arg1689 generates a 70-kDa A3-C1-C2 subunit. Analyses of mutations and hemophilia A database suggest that proteolysis at the Arg372 and Arg1689 sites is essential for generating Factor VIIIa cofactor activity (Non-Patent Document 7). Cleavage at the former site exposes a functional Factor IXa-interactive site within the A2 domain which is cryptic in the inactivated molecule (Non-Patent Document 8). Meanwhile, cleavage at the latter site liberates the cofactor from its carrier protein, vWF, and contributes to the overall specific activity of the cofactor (Non-Patent Documents 9 and 10).
Serine proteases including activated protein C (APC), Factor Xa, Factor IXa, and plasmin inactivate Factor VIII (a) by cleavage at Arg336 in the A1 domain. The inactivation by cleavage at Arg336 can be associated with altered interaction between the A2 subunit and truncated A1, and the increase of Km for the substrate Factor X. The latter reflects the loss of a Factor X-interactive site within residues 337 to 372 of A1. Furthermore, Factor Xa and APC also attack the Lys36 and Arg562 sites, respectively. Cleavage at these sites has been suggested to change the structure of A1. The structural change of A1 limits its productive interaction with the A2 subunit, and impairs its bonding with Factor VIIIa A2 subunit and Factor IXa molecule in the Xase complex.
Factor VIII inhibitors are generated as isoantibodies in 20 to 30% of multi-transfused hemophilia A patients. Furthermore, autoantibodies can also arise in individuals who are originally normal.
In general, most of the Factor VIII inhibitor isoantibodies and autoantibodies that function as an anti-Factor VIII neutralizing antibody reduce or eliminate the Factor VIII activity. The Factor VIII-neutralizing mechanism of such antibodies has been intensively studied by various researchers. Antibodies that recognize one or more of the A2, C2, or A3-C1 epitopes block the binding of the Factor VIII molecule to some coagulation factors, for example, vWF (Non-Patent Documents 12 and 13), phospholipid (Non-Patent Documents 13 and 14), and Factor IXa (Non-Patent Documents 18 and 19). Furthermore, some of the antibodies inhibit the activation of Factor VIII mediated by thrombin (Non-Patent Document 14) or Factor Xa (Non-Patent Documents 15 and 16). Meanwhile, anti-Factor VIII antibodies that do not have the ability to neutralize Factor VIII activity have also been reported (namely, non-neutralizing antibodies) (Non-Patent Documents 20 and 21). Such antibodies in normal individuals or hemophilia A patients can be confirmed only by ELISA. However, most of the non-neutralizing antibodies are not expected to have any significant function, and the epitopes recognized by these antibodies remained unidentified.
Furthermore, for the treatment of blood coagulation-related diseases such as hemophilia A, it has been proposed to use antibodies that inhibit the binding between Factor VIII and low density lipoprotein receptor protein (LRP) which inactivates Factor VIII by binding to the A2 domain of activated blood coagulation factor VIII (Patent Document 1). In addition, antibodies that inhibit the reaction between Factor VIII and APC have been reportedly found in hemophilia A patients (Non-Patent Document 22). However, there is no known antibody to date that has the function of enhancing the activation of blood coagulation factor VIII.    [Patent Document 1] WO03/093313    [Non-Patent Document 1] Mann K. G., Nesheim M. E., Church W. R., Haley P., and Krishnaswamy S. (1990) Blood 76, 1-16    [Non-Patent Document 2] Hoyer L. W. (1981) Blood 58, 1-13    [Non-Patent Document 3] Wood W. I., Capon D. J., Simonsen C. C., Eaton D. L., Gitschier J., Keyt B., Seeburg P. H., Smith D. H., Hollingshead P., Wion K. L., Delwart E., Tuddenham E. D. G., Vehar G. A., and Lawn R. M. (1984) Nature 312, 330-7    [Non-Patent Document 4] Vehar G. A., Keyt B., Eaton D., Rodriguez H., O'Brien D. P., Rotblat F., Oppermann H., Keck R., Wood W. I., Harkins R. N., Tuddenham E. G. D., Lawn R. M., and Capon D. J. (1984) Nature 312, 337-42    [Non-Patent Document 5] Fay P. J., Anderson M. T., Chavin S. I., and Marder V. J. (1986) Biochim. Biophys. Acta 871, 268-78    [Non-Patent Document 6] Eaton D., Rodriguez H., and Vehar G. A. (1986) Biochemistry 25, 505-12    [Non-Patent Document 7] Fay P. J. (2004) Blood Rev. 18, 1-15    [Non-Patent Document 8] Fay P. J., Mastri M., Koszelak M. E., and Wakabayashi H. (2001) J. Biol. Chem. 276, 12434-9    [Non-Patent Document 9] Lollar P., Hill-Eubanks D. C., and Parker C. G. (1988) J. Biol. Chem. 263, 10451-5    [Non-Patent Document 10] Regan L. M., and Fay P. J. (1995) J. Biol. Chem. 270, 8546-52    [Non-Patent Document 11] Shima M. (2006) Int. J. Hematol. 83, 109-18    [Non-Patent Document 12] Shima M., Nakai H., Scandella D., Tanaka I., Sawamoto Y, Kamisue S., Morichika S., Murakami T., and Yoshioka A. (1995) Br. J. Haematol. 91, 714-21    [Non-Patent Document 13] Shima M., Scandella D., Yoshioka A., Nakai H., Tanaka I., Kamisue S., Terada S., and Fukui H. (1993) Thromb. Haemostasis 67, 240-6    [Non-Patent Document 14] Scandella D., Gilbert G. E., Shima M., Nakai H., Eagleson C., Felch M., Prescott R., Rajalakshmi K. J., Hoyer L. W., and Saenko E. (1995) Blood 86, 1811-9    [Non-Patent Document 15] Nogami K., Shima M., Hosokawa K., Nagata M., Koide T., Saenko E. L., Tanaka I., Shibata M., and Yoshioka A. (2000) J. Biol. Chem. 275, 25774-80    [Non-Patent Document 16] Nogami K., Shima M., Hosokawa K., Suzuki T., Koide T., Saenko E. L., Scandella D., Shibata M., Kamisue S., Tanaka I., and Yoshioka A. (1999) J. Biol. Chem. 274, 31000-7    [Non-Patent Document 17] Nogami K., Shima M., Nishiya K., Sakurai Y., Tanaka I., Giddings J. C., Saenko E. L., and Yoshioka A. (2002) Thromb. Haemostasis 87, 459-65    [Non-Patent Document 18] Fay P. J., and Scandella D. (1999) J. Biol. Chem. 274, 29826-30    [Non-Patent Document 19] Zhong D., Saenko E. L., Shima M., Felch M., and Scandella D. (1998) Blood 92, 136-42    [Non-Patent Document 20] Batle J., Gomez E., Rendal E., Torea J., Loures E., Couselo M., Vila P., Sedano C., Tusell X., Magallon M., Quintana M., Gonzalez-Boullosa R., and Lopes-Fernandez M. F. (1996) Ann Hematol 72, 321-6    [Non-Patent Document 21] Blanco A. N., Peirano A. A., Grosso S. H., Gennari L. C., Bianco R. P., and Lazzari M. A. (2000) Haematologica 85, 1045-50    [Non-Patent Document 22] Nogami K., Shima M., Giddings J. C., Hosokawa K., Nagata M., Kamisue S., Suzuki H, Shibata M., Saenko E. L., Tanaka I., and Yoshioka A. (2001) Blood 97, 669-77